Natural Products as Antibacterial Agents—Antibacterial Potential and Safety of Post-distillation and

31 Pages·2017·0.96 MB·English

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Chapter 6
Natural Products as Antibacterial Agents — Antibacterial
Potential and Safety of Post-distillation and Waste
Material from Thymus vulgaris L., Lamiaceae
Neda Gavarić, Jasna Kovač, Nadine Kretschmer,
Nebojša Kladar, Sonja Smole Možina, Franz Bucar,
Rudolf Bauer and Biljana Božin
Additional information is available at the end of the chapter
http://dx.doi.org/10.5772/60869
Abstract
Medicinal plants have a long tradition of use in folk and conventional medicine. In
recent years numerous studies confirm various bioactivities of natural products,
among them antibacterial activity. Natural antibacterial agents such are essential oils
and isolated compounds now represent a notable source for pharmaceutical and food
industry and are widely used in cosmetology. They meet standards of 'green
consumerism' together with excellent antibacterial activity. Aromatic plants such is
Thymus vulgaris L. are the major sources of essential oils. Thyme essential oil, as well
as dominant compounds thymol and carvacrol are generally recognised as safe and
have been registered by European Commission for use as flavouring agents in
foodstuffs. However, essential oil is present in very low amount (0,8-2,6%) in thyme
leaves. Thus, the majority of plant material remains unused after the isolation.
Nowadays, the biological potential of various plant waste materials are in focus of
numerous studies. These investigations also include the antimicrobial activity
considering the fact that waste material extracts represent the valuable source of
different phenolic compounds. Regarding all this, the aim of the present study was to
determine antibacterial potential of chemically characterised extracts obtained from
waste material remaining after the preparation of drug (stems) and isolation of thyme
essential oil (deodorised leaves, postdistillation decoction) on selected bacterial
strains. Also, in order to determine safety of waste extracts their cytotoxicity was
investigated. All extracts were prepared with maceration using 45% or 75% ethanol
© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,
and reproduction in any medium, provided the original work is properly cited.
124 Concepts, Compounds and the Alternatives of Antibacterials
(EtOH) for 24 h at room temperature (1:10 w/v). Total phenolic compounds and
flavonoids were determined spectrophotometrically. Extracts were chemically
characterized by HPLC/DAD analysis. Antibacerial testing was done with broth
dilution method against several bacterial strains (Staphylococcus aureus, Bacillus cereus,
Salmonella infantis, Escherichia coli and Campylobacter jejuni). Cytotoxicity and cytopro‐
tection studies were performed by XTT assay. Result of HPLC analysis showed that
investigated extracts, especially those obtained from deodorised leaves represent a
valuable source of rosmarinic acid and luteolin 7-O-glucuronide. Antibacterial testing
indicated that all waste material extracts, except the extract T2, possess similar or even
stronger bacteriostatic activity than T1. No cytotoxicity nor cytoprotection were
determined. In conclusion, results of this study confirmed antibacterial potential
investigated thyme extracts. High concentrations of rosmarinic acid and luteolin 7-O-
glucuronide, which both have numerous pharmacological activities, were deter‐
mined. This indicates that thyme postdistillation waste material extracts could be used
for isolation of dominant compounds or as addities in pharmaceutical and food
industry.
Keywords: aromatic plants, thyme, postdistillation waste, antibacterial potential,
cytotoxicity
1. Introduction
Plants have been used as food, spice, and medicine since ancient time. Much before the
discovery of the existence of microorganisms, plants were an integrated part of the traditional
medicine of many communities used for the treatment of various infectious diseases. Likewise,
the use of plants as spices, not only affected the organoleptic properties of food, but it was also
one of the ways for its preservation [1]. Today, modern consumers demand food that will be
minimally processed and contain, as much as possible, additives of natural origin that unlike
synthetic ones, will be safe and ecologically acceptable. Also, the approach in raising livestock
and production of food of plant origin is shifting towards the use of traditional methods in
protection against pests and diseases [2]. In addition, the irrational use of antibiotics not only
in human and veterinary medicine, but also to promote growth in agriculture, has led to more
frequent occurrence of resistance to conventional antibiotics of some pathogenic microorgan‐
isms [3, 4, 5] such are Escherichia coli, Staphylococcus aureus, Enterococcus spp., Salmonella spp.,
and Campylobacter spp. [6, 7, 8, 9]. Moreover, it is important to bear in mind the potential
synergistic action of different natural products and conventional antibiotics in order to affect
the emerging resistance [10, 11, 12].
Aromatic plants such as Thymus vulgaris L. (common thyme) and many other representatives
of the family Lamiaceae have a long tradition of use in both folk and conventional medicine
and in the pharmaceutical industry [13]. Common thyme is indigenous to Europe, especially
the Mediterranean region, and is extensively cultivated worldwide. It is a small, bushy herb,
Natural Products as Antibacterial Agents — Antibacterial Potential and Safety of Post-distillation and Waste… 125
http://dx.doi.org/10.5772/60869
with small, elliptical, greenish-grey, and shortly-stalked leaves. Thyme has a characteristic
odour of thymol and is used as a culinary herb [14]. The active principle is the essential oil
(Thymi aetheroleum), with thymol (25%–50%) and carvacrol (3%–10%) as dominant compounds
(Table 1) (Figure 1) [15].
Compounds Percentage (%) References
Monoterpene Hydrocarbons
α-Terpinene 1.25–3.17 17
p-Cimene 0.8–16.13 16, 17
γ-Terpinene 7.3–1.87 12, 16, 17
Oxygenated Monoterpenes
1, 8-Cineole 0.8–2.17 16, 17
Linalool 2.03–6.8 12, 16, 17
Menthone 2.2 16
Borneol 2.6–8.9 12, 16
Neomenthol 2.8 16
Terpinen-4-ol 1–2.63 16, 17
Aromatic Oxygenated Monoterpenes
Carvacrol methyl ether 3.9 12
Thymol 35.51–47.9 12, 16, 17
Carvacrol 4.43–12
Sesquiterpene Hydrocarbons
trans-Caryophyllene 1.13–5.1 12, 17
β-Cubebene 2.4 16
Oxygenated Sesquiterpenes
Caryophyllene oxide 0.33–4.6 12, 17
Aliphatic compounds
1-Octen-3-ol 2.8 12
Table 1. Dominant compounds of Thymus vulgaris essential oil
The percentage of the major constituents varies greatly, depending on numerous complex
factors, both endogenous and exogenous such are: chemotype, ontogenesis, geographic and
climatic conditions, methods used for plant material processing, and essential oil isolation [18].
Also, similar to other medicinal plants, the drug could be adulterated or replaced with other
representatives of the genus Thymus, such as T. serpyllum, T. marschallianus, T. pannonicus, etc.,
that affect the differences in chemical composition and biological activities [19, 20]. Neverthe‐
less, a minimum 40% of the thyme essential oil is required to be aromatic oxygenated mono‐
terpenes [15].
126 Concepts, Compounds and the Alternatives of Antibacterials
Thyme essential oil Thymol Carvacrol References
(µl/mL) (µg/mL) (µg/mL)
Staphylococcus aureus 0.2–2.5 250–2000 125–280 12, 21, 22
Salmonella sp. 0.45–20 250–3755 125–375 3, 12, 21
Escherichia coli 0.45–1.25 250–2000 125–560 12, 21, 22, 23
Bacillus cereus 1 250–560 125–140 12, 21
Table 2. Minimal inhibitory concentrations of thyme essential oil, thymol, and carvacrol on selected bacterial strains
Thyme essential oil, thymol, and carvacrol are generally recognised as safe (GRAS status) and
have been registered by the European Commission for use as flavouring agents in foods [24].
GRAS is a rigorous process that relies on common knowledge and expert consensus about the
safety of the substance for its intended use. Well-conducted toxicology studies are one of the
factors that are used to assess the safety of various natural products, for example, plant extracts
or essential oils that are composed of a mixture of tens or hundreds of compounds [25].

Thymol  Carvacrol 
Figure 1. Chemical structures of dominant monoterpenes in thyme essential oil
In recent years, numerous studies confirm various bioactivities of thyme essential oil and
isolated dominant monoterpenes, among them antibacterial and antifungal activity, together
with the inhibition of mycotoxin production (Table 2) [3, 12, 16, 21, 23, 26, 27, 28]. Generally,
most of the essential oils used in the pharmaceutical, cosmetic, and food industries possess
strong antioxidant activity and represent a notable source of natural additives for different
products [12, 29, 30]. What is more important is that they meet standards of 'green consumer‐
ism' [21]. However, the essential oil is present in very low amounts (0.8%–2.6%) in thyme
leaves. Thus, the majority of plant material remains unused after the isolation.
On the other hand, thyme leaves are found to be a rich source of various phenolic compounds,
particularly phenolic acids, flavonoids, and other secondary metabolites that are expected to
contribute to its biological activities (Table 3) [14].
Rosmarinic acid is an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid (Figure 5) widely
found in the plant kingdom and presumably accumulated as a defense compound. In the
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Lamiaceae family, the occurrence is mainly restricted to the subfamily Nepetoideae [31] and
it is identified as one of the active components of several aromatic medicinal plants (e.g., Salvia
officinalis, Mentha x piperita, Lavandula officinalis, Ocimum basilicum, Origanum vulgare, Origanum
majorana, Thymus vulgaris, Melissa officinalis, Rosmarinus officinalis) [32]. For rosmarinic acid
numerous and notable biological activities are confirmed in different in vitro and in vivo
examinations. It is of pharmaceutical importance because of its non-specific complement
activation and inhibition of the biosynthesis of leukotriens (leading to an anti-inflammatory
effect), as well as its antimicrobial and especially antiviral activity [14, 33]. Additionally, it
exhibits a very strong antioxidant effect; it is an inhibitor of acetyl and butyryl cholinesterase
and neuroprotector that propose its use in the prevention and symptomatic treatment of
Alzheimer's disease. However, it should be considered that bioavailability of rosmarinic acid
in the brain is very unlikely. Also, in some test systems rosmarinic acid expresses anticarci‐
nogenic activity [34, 35, 36].

Quercetin  Rutin 

Figure 2. Chemical structures of flavonoids in investigated thyme extracts (Glu = glucose, Rha = rhamnose)
Extraction procedure Identified compounds References
Butanol-soluble fraction Acetophenone glycosides (Androsin, Picein, glycosides of 4-[37]
hydroxyacetophenone derivatives)
Methanol, reflux, residue was Acidic polysaccharide [38]
extracted with distilled water at
100°C for 2 h
Acetone using a Polytron Caffeic acid, Luteolin, Rosmarinic acid, Hispidulin [39]
homogenizer for 1 min
CHCl soluble fraction 5,4`-Dihydroxy-6,7,8-trimethoxyflavone, 5,3`,4`- [40]
2 2
Trihydroxy-7-methoxyflavone, 5,4`-Dihydroxy-6,7,3`-
trimethoxyflavone, 5,4`-Dihydroxy-6,7,8,3`-
tetramethoxyflavone
β
Extracted at 20°C with 1L of EtOH- Roβsmarinic acid, 3`-O-(8``-Z-Caffeoyl) rosmarinic aciβd, [41]
HO-HOAc (80:19:1) under N for 5 Eriodictyol, Taxifolin, Luteoline-7-O-glucuronide
2 2
days; further fractionation
128 Concepts, Compounds and the Alternatives of Antibacterials
Extraction procedure Identified compounds References
Methanol; 1 day at room temperaturebiphenyl compounds (e.g. 3,4,3`,4`-tetrahydroxy-5,5`- [42]
diisopropyl-2,2`-dimethylbiphenyl)
Deodorised aqueous extracts Total phenolics (mg/g gallic acid equivalents) [43]
Methanol extract Monoterpene glucosides (p-Cymenol-9-O-β-glucoside, 2- [44]
and 5-O-β-glucosides of thymoquinol, angelicoidenol-O-β-
glucoside); Arbutin
Ethanol/water (30:70, v/v) with the Rosmarinic acid, Caffeic acid [45]
aid of sonication for 10 min
methanol extract compounds related to hydroxyjasmone (e.g. 5- [46]
hydroxyjasmone-5-O-ß-glucoside) and simple phenol
glucosides (3-Hydroxy-4-methoxyphenethyl-3-O-ß-
glucoside, eugenol-O-ß-glucoside, syringin)
Methanol, 20 min, 60°C in a water Luteolin-glucoside, Rosmarinic acid, Eriodictyol, Luteolin, [47]
bath with shaking Apigenin
Methanol, 6 h, Soxhlet apparatus Oleanolic acid, Ursolic acid [48]
aqueous extracts (boiling water), 30 Luteolin-7-O-glucuronide, Lithospermic acid, Rosmarinic [49]
min acid, Caffeic acid
Aquous, aq. methanolic, methanolic Arbutin, Hydroquinone, Naringenin, Naringenin-7-O-ß- [50]
(1:100 or 1:200); infusion, hot glucoside, Narirutin, Eriodictyol, Eriodictyol-7-O-ß-
methanolic, usage of ultrasonic bath glucoside, Eriocitrin, Hesperidin, Luteolin, Luteolin-7-O-ß-
for 15 or 30 min glucuronide, Luteolin-7-O-ß-rutinoside, Rosmarinic acid,
Methyl rosmarinate, Caffeic acid
95% ethanol at 60°C for 2 h with Total phenolics (mg/g sinapic acid equivalents) [51]
stirring
Liquid nitrogen powdered samples Caffeic acid, Rosmarinic acid [52]
were extracted with acidified
methanol
Table 3. Comparison of literature data on identified compounds in Thymus vulgaris leaves
Chlorogenic acid (5-O-caffeoylquinic acid) is a natural antioxidant that is produced in various
plants as a response to pathogen microorganisms, different mechanical impairment, or
excessive exposure to light (Figure 5) [53]. Due to these characteristics, chlorogenic acid found
its use in pharmaceutical and food industries, as well as in cosmetology [54]. Gallic and ferulic
acids are also proven antioxidant agents (Figure 3) [45].
Furthermore, many beneficial properties have been identified for flavonoids (e.g., quecetin
and rutin as the most widely distributed in nature) (Figure 2), and one of the most popularly
cited property is their antioxidant activity. Other actions that are proposed to contribute to
their biological effects include chelating metal ions, stimulating phase II detoxifying enzyme
activity, and inhibiting proliferation and inducing apoptosis. Also, for some of the flavonoids
Natural Products as Antibacterial Agents — Antibacterial Potential and Safety of Post-distillation and Waste… 129
http://dx.doi.org/10.5772/60869

Gallic acid  Protocatechuic acid  Syringic acid 

Caffeic acid  p‐Coumaric acid  Ferulic acid 

Figure 3. Chemical structures of hydroxybenzoic and hydroxycinnamic acids present in thyme extracts
where anti-inflammatory activity has been confirmed, they decrease the vascular cell adhesion
and molecule expression, increase endothelial nitric oxide synthase activity, as well as inhibit
platelet aggregation [55].
Specifically, it is determined that luteolin (3',4',5,7-tetrahydroxyflavone) exhibit notable
antioxidant, antimicrobial, and anti-inflammatory activities [56]. Also, there is some evidence
that apigenin and luteolin act as antimutagenic and anticarcinogenic agents (Figure 4) [57].

Apigenin  Luteolin   

Figure 4. Chemical structures of dominant flavones in investigated thyme extracts
Since various plant extracts and isolated natural compounds are widely used and represe nt
one of the major approaches in alternative and complementary medicine that are also accepted
by conventional medicine, their quality, efficacy, and safety are of the utmost importance [58].
Safety issues are especially significant due to increasing body of evidence on side effects and
130 Concepts, Compounds and the Alternatives of Antibacterials

interactions between herbal remedies or/and food or conventional drugs. These interactions
sometimes can be very serious. They may lead to increased or lack of action of conventional
drugs and caution must be taken in the application of plant extracts [59].

Nowadays, the biological potential of various plant waste materials is the focus of numerous
studies. This trend includes not only the examination of aromatic plants [43, 60, 61, 62], but
also different fruits and plant products such as wine, olives, beetroot, tomato, garlic, and
pomegranate [63, 64, 65, 66]. These investigations also include their antimicrobial activity,
considering the fact that waste material extracts represent the valuable source of different
phenolic compounds. These studies are important, both in terms of economy and ecology, in
order to elucidate the way to exploit post-distillation waste material of aromatic plants and
other plant waste materials more efficiently.

Chlorogenic acid 
Rosmarinic acid 

Figure 5. Chemical structures of dominant caffeic acid oligomers in thyme extracts
The aim of the present study was to determine the antibacterial potential of chemically well-
characterised extracts obtained from waste material that remain after the preparation of the
drug Thymi folium (stems) and the isolation of thyme essential oil (deodorised leaves and post-
distillation decoction) on selected bacterial strains. Also, in order to determine the safety of the
waste extracts, their cytotoxicity was investigated.
2. Methods
2.1. Plant material and extract preparation
In July 2013, aboveground parts of thyme (Thymus vulgaris L., Lamiaceae) were collected, just
before flowering, in Padej, the Vojvodina province, Republic of Serbia. Voucher specimen of
Natural Products as Antibacterial Agents — Antibacterial Potential and Safety of Post-distillation and Waste… 131
http://dx.doi.org/10.5772/60869
collected plants (no. ThV-15/10) [67] was confirmed and deposited at the Herbarium of the
Laboratory of Pharmacognosy, Department of Pharmacy, Faculty of Medicine, University of
Novi Sad. Leaves were separated from the previously air-dried stems and stored separately
at a cool and dry place for extract preparation. Standard thyme leaves extract [15, 68] was
prepared by maceration procedure in 45% ethanol (EtOH) as a solvent for 24 h (1:10 w/v, 10 g
dried leaves) at room temperature (T1). Essential oil was isolated using hydrodistillation
technique [69]. The waste material, remaining after isolation, was filtered and obtained
decoction was used to prepare post-distillation extract (T2). Deodorised leaves were air dried
and macerated with 45% (T3) and 75% (T4) EtOH for 24 h. Dried stems were grinded (sieve
0.75) and macerated with 45% EtOH for 24 h to obtain the extract T5.
After the maceration, extracts were collected, filtered, and evaporated to dryness under
vacuum. The quantities of dry extracts were determined gravimetrically. Residues were
dissolved in water to make 10% (w/v) stock solutions for further investigation. For the high-
performance liquid chromatography (HPLC analysis), residues were dissolved in methanol,
1% formic acid mixture (50:50 v/v) to make 2% (w/v) stock solutions.
2.2. Chemical composition
2.2.1. Determination of total phenolic compounds and total flavonoid content
The amount of total phenolic compounds in investigated extracts was determined spectro‐
photometrically with Folin-Ciocalteu (FC) reagent [70]. The concentration of total phenolic
compounds was expressed in mg of gallic acid equivalents (GAE) per g of dry extract (d.e.),
using a standard curve of gallic acid (concentration range 0.08–0.24 mg/mL). Total flavonoid
content was also determined spectrophotometrically using a method based on the formation
of a flavonoid-aluminium complex with an absorptivity maximum at 430 nm [71]. Flavonoid
content was expressed in mg of quercetin equivalents (QE) per g of dry extract, using a
standard curve of quercetin (concentration range 10–100 μg/mL). All measurements were done
in triplicate.
2.2.2. HPLC/DAD analysis
Investigated thyme leaves extracts are rich in a wide range of phenolic compounds [50].
Therefore, a method that allows simultaneous detection of various phenolics in a single run
was developed. This decreases the time necessary for analysis and reduces the analysis costs
[72]. HPLC analysis was performed using a liquid chromatograph (Agilent 1200 series),
equipped with diode array detector (DAD) and Eclipse XDB-C18, 1.8 μm, 4.6×50 mm column,
at a flow-rate of 1 mL/min. Solvent gradient was performed by varying the proportion of
solvent A (methanol) to solvent B (1% formic acid inwater (v/v)) [73]. The total running time
and post-running time were 45 and 10 min, respectively. The column temperature was 30ºC.
The injected volume of samples and standards was 5 μL and it was done automatically using
an autosampler. The spectra were acquired in the range of 210–400 nm and chromatograms
plotted at 280, 330, and 350 nm with a bandwidth of 4 nm, and with reference wavelength/
bandwidth of 500/100 nm. Quantification of selected phenolics: gallic acid, protocatechuic acid,
caffeic acid, chlorogenic acid, syringic acid, p-coumaric acid, ferulic acid, rutin, rosmarinic acid,